Epithelial cells line the surfaces of our organs and are critical for their formation and function. During embryogenesis most epithelial cells develop when mesenchymal cells polarize along their apicobasal axis and assemble cell-cell junctions (mesenchymal-to-epithelial transformation, MET). Loss of polarity or junctions can lead to devastating diseases, including kidney disease, cancer caused by increased cell proliferation and invasion, and birth defects arising from impaired morphogenesis. Much of our understanding of MET comes from in vitro studies of cultured cells, which polarize as they make E-cadherin-mediated contact with one another. However, mechanisms of MET likely differ in vivo since E-cadherin is not needed to initiate polarity in Drosophila, C. elegans, and some mammalian epithelia, and because cell contact is not always sufficient to induce polarization. The long-term goal of this project is to determine the cellular and molecular mechanisms used to polarize epithelial cells and assemble junctions in a developing organism. We have developed live-imaging and genetic tools to investigate mechanisms of MET in living C. elegans embryos. Using these tools we have shown that conserved polarity regulators induce junction formation in sequential steps. First, PAR-3 establishes polarity by aggregating junction proteins and other polarity regulators into cortical foci, which then travel to the apical surface. Then PAR-6, which functions with the kinase PKC-3, condenses clusters of apical junction proteins into belts that encircle the cell. We have also shown that polarization mechanisms can differ between tube-forming epithelial cells and sheet-forming epithelial cells, where the apical Crumbs protein EAT-20 functions redundantly with PAR-3 to polarize cells. Although PAR-3, EAT-20, PAR-6, and PKC-3 homologues have conserved roles in epithelial cells, how these proteins function to polarize cells and assemble junctions in vivo is largely unknown. The specific objectives of the proposed research are to define the molecular mechanisms that PAR-3 and EAT-20 use to polarize epithelial cells, and to determine how PAR-6 and PKC-3 assemble junctions. Using live imaging and genetic analysis, we will determine how PAR-3 foci form, how junction and polarity proteins load onto foci, and test the hypothesis that foci move to the apical surface along microtubules. We will test the hypothesis that EAT-20 establishes polarity by recruiting proteins to the apical surface, define the pathways downstream of EAT-20 that polarize cells, and determine if EAT-20 apical localization is directed by cell contacts or extra- embryonic ligands. Finally, we will use large-scale genetic selection screens we have already performed to identify genes that regulate or function downstream of PAR-6 and PKC-3 to assemble junctions. This proposal will advance the field by establishing mechanisms epithelial cells use to polarize and assemble junctions in vivo, increasing our understanding of epithelial diseases including kidney disease, cancer, and birth defects.